(Poly)Aminoalkylaminoalkylamide, alkyl-urea, or alkyl-sulfonamide derivatives of epipodophyllotoxin, a process for preparing them, and application thereof in therapy as anticancer agents

ABSTRACT

The present invention relates to new derivatives of epipodophyllotoxin 4-substituted with an optionally substituted (poly)aminoalkylaminoalkylamide, or alkyl-urea or alkyl-sulfonamide chain, a process for preparing them and their use as a medicine as an anticancer agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 12/737,645filed on Feb. 2, 2011, now U.S. Pat. No. 8,288,567, which is a NationalPhase of PCT International Application No. PCT/EP2009/060739 filed onAug. 19, 2009, which claims the benefit of Patent Application No.0855629 filed in FRANCE, on Aug. 19, 2008. The entire contents of all ofthe above applications is hereby incorporated by reference.

The present invention relates to new derivatives of podophyllotoxin4-substituted with an optionally substituted(poly)aminoalkylaminoalkylamide or alkyl-sulfonamide or alkyl-ureachain, a process for preparing them and their use as a medicine, inparticular as anticancer agents.

The compounds of the present invention are derived from podophyllotoxin,a natural lignane known as a therapeutic agent in the treatment ofcancer. Other synthetic derivatives such as etoposide or teniposide arepart of the therapeutic arsenal for the treatment of small cell lungcancer in particular. These various compounds act by inhibiting thecatalyst activity of topoisomerase II.

The alkylamine substitution in the 4β-position of the4′-demethylpodophyllotoxin backbone thus represents a spermine orspermidine alkylamide unit, or more generally a(poly)aminoalkylaminoalkylamide unit. Likewise this substitutionrepresents a spermine or spermidine alkyl-sulfonamide unit, or moregenerally a (poly)aminoalkyaminoalkylsulfonamide unit. Further, thissubstitution represents a spermine or spermidine alkylurea unit, or moregenerally a (poly)aminoalkylaminoalkylurea unit.

The 4′-demethylepipodophyllotoxin derivatives are known as2-topoisomerase inhibitors. Their cytotoxic and antitumor activitieshave been discovered and revealed, in particular with etoposide, TOP 53(Drugs of the Future 1996, 21, 1136), GL 331 (Medicinal ResearchReviews, 1997, 17, 367), and NK 611 (Cancer Chemother. Pharmacol. 1996,38, 217 and 541). Compounds having benzylamine-type amine chainsdirectly linked to the 4β-position of the podophyllotoxin have beendescribed (J. Med. Chem. 1991, 34, 3346). Patent application FR 2 810321 discloses carbamate- or thiocarbamate-type podophyllotoxinderivatives useful in the treatment of cancer. Amide compounds in the4β-position have also been described (U.S. Pat. No. 6,566,393; ActaPharmaceutica Sinica (Yaoxue Xuebao), 1993, 28, 422; Acta Chem. Scand.1993, 47, 1190; Anti-Cancer Drug Design 2001, 16, 305). Urea compoundsin the 4β-position have been described (Heterocycles 1994, 39, (1), 361;J. Med. Chem. 2002, 45, 2294).

The patent EP 0 876 374 discloses a process for demethylatingpodophyllotoxin and readily gives the 4′-demethylepipodophyllotoxin,which is a synthesis intermediate in the preparation of etoposide andteniposide.

The international application WO 03/082876 discloses 4β-4″-[{2″-benzoylsubstituted}anilino] derivatives of podophyllotoxin having an anticanceractivity.

The need to provide more effective treatments encourages the search fornew molecules having various mechanisms of action, thereby targetingcurrently badly treated or non-treated types of tumors, as well asavoiding resistance problems. The availability of these new productsalso allows to develop protocols including co-treatments, which are moreactive on some tumors.

The new compounds of the present invention provide a way to overcomethis problem.

Compounds described in patent application WO 2005/100363 have anacetamide moiety in the 4β-position of the podophyllotoxin unit, saidmoiety being linked to an amine or polyamine chain. We have synthesizedother derivatives having an alkylamide, urea or sulfonamide moiety anddocumented their cytotoxic and anticancer activity.

The present invention relates to compounds of the general formula 1:

whereinR represents hydrogen or C₁₋₄alkyl,A represents CO(CH₂)_(n), where n=2, 3, 4, or 5, orA represents CONH(CH₂)_(n), where n has the same values as describedabove, orA represents SO₂(CH₂)_(n), where n has the same values as describedabove,R1=H, or C₁₋₄alkyl,R2=H, or C₁₋₄alkyl, orR2 can also be (CH₂)_(m)—NR3R4, where R3=H, or C₁₋₄alkyl, and m=2, 3, 4,or 5,R4=H, or C₁₋₄alkyl, orR4 can also be (CH₂)_(p)—NR5R6, where R5=H, or C₁₋₄alkyl, and p=2, 3, 4,or 5, andR6=H, or C₁₋₄alkyl, orR6 can also be (CH₂)_(q)—NH₂, where q=2, 3, 4, or 5,with the exception of the compound wherein A=CO(CH₂)₂ where R1=R2=H.

The term <<C₁₋₄alkyl>>, as defined in the present invention, is meant torefer to a saturated, linear or branched, hydrocarbon chain comprising 1to 4 carbon atoms. An example includes methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, and tert-butyl groups. Thus, throughout thisspecification, C₃ and C₄ alkyl groups designate both linear and branchedgroups.

The invention also relates to their salts, particularly theirpharmaceutically acceptable water-soluble salts, especially inorganic ororganic acid addition salts thereof, as well as pharmaceuticalcompositions containing them, and their use as a medicine, in particularintended for treating cancer.

Urea, amide, carbamate or sulfonamide podophyllotoxin derivatives havebeen described in the literature and in patents (Zhongguo Yaoke DaxueXuebao 1993, 24, 134; WO 2004/000859; US 2004/0106676; J. Med. Chem.2004, 47, 2365; Org. Biomol. Chem. 2005, 3, 1074; WO 2004/073375;Bioorg. Med. Chem. 2003, 11, 5135). Their activity indicates aninhibiting action on 2-topoisomerase and a value as a compound having anantitumor activity. However the low water-solubility of these compoundsmakes them difficult to use. While a basic nitrogen atom present in themolecule makes it optionally possible to prepare a soluble salt, it isnot always obvious to achieve an active compound having the requiredantitumor properties.

The literature does not disclose any compound having a polyamine chaingrafted in the 4β-position of the 4′-demethyl-4-deoxypodophyllotoxin,through a spacer, with the exception of patent application WO2005/100363.

Accordingly the present invention discloses new polyamine derivativesderived from podophyllotoxin.

The compounds of the present invention have an epipodophyllotoxinstructure substituted in the 4β-position with a urea unit linked to apolyamine chain such as in particular putrescine, spermine orspermidine, but also other polyamines. Likewise this 4β-position can belinked to an amide group, whether the latter is linked or not to alinear spacer having 2 to 5 carbon atoms then to a polyamine such asputrescine, spermine or spermidine or other polyamines.

The 4β-position can be also substituted with a sulfamidoethyl unitlinked in turn to a polyamine chain such as putrescine, spermine orspermidine. The polyamine transportation system has already beenutilized for targeting cytotoxic polyamine analogs (Annu. Rev.Pharmacol. Toxicol. 1995, 35, 55; Medicine/Sciences 1996, 12, 745), butseemingly without success.

Compounds having a polyamine chain grafted on a DNA intercalating unitof the acridine (J. Org. Chem. 2000, 65, 5590; J. Med. Chem. 2002, 45,5098), or indenoisoquinoline (J. Med. Chem. 2003, 46, 5712) type havebeen described.

The property of the compounds of the present invention is that they areDNA targeting agents, and successfully induce damages within said DNA,both qualitatively and quantitatively different from the other knownanticancer compounds such as etoposide.

The presence of a polyamine chain, such as for example putrescine,spermine or spermidine, is advantageous in that it is recognized by thetransportation system of natural polyamines used by the cancer cell toproliferate (J. Cell. Physiol. 1993, 155, 399; Annu. Rev. Biochem. 1984,53, 749). This provides the compounds of the present invention with apreferred passage way towards the cancer cell preferentially comparedwith other cells. The compounds of the present invention thus have invitro cytotoxic properties and in vivo antitumor properties.

Furthermore, an important advantage of these compounds is the presenceof amine functions providing a good water solubility, which makes themconvenient in terms of formulation, administration, dispensing ability,and bioavailability in the body. The pharmacokinetic parameters are thusimproved.

The preferred compounds of the invention are selected from the followingcompounds:

Amide Series:

Compound 1:3-(2-Dimethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 2:4-(2-Dimethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 3:3-[(2-Dimethylaminoethyl)-methylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 4:4-[(2-Dimethylaminoethyl)-methylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 5:3-Dimethylamino-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 6:4-Dimethylamino-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 7: 5-Dimethylaminopentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 8:3-(2-Diethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 9:4-(2-Diethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 10:3-(2-Diethylaminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 11:4-(2-Diethylaminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 12:3-(2-Aminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 13:3-(3-Aminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 14:3-(4-Aminobutylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 15:4-(3-Aminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 16:4-(4-Aminobutylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 17: 5-(4-Aminobutylamino)pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]-dioxol-5-yl]-amide

Compound 18:3-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 19:3-{3-[3-(3-Aminopropylamino)-propylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 20:3-{4-[4-(4-Aminobutylamino)-butylamino]-butylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 21:4-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 22:4-{3-[3-(3-Aminopropylamino)-propylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 23:4-{4-[4-(4-Aminobutylamino)-butylamino]-butylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 24:5-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 25:5-{3-[3-(3-Aminopropylamino)-propylamino]-propylamino}pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 26:5-{4-[4-(4-Aminobutylamino)-butylamino]-butylamino}pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 27:3-[3-(4-Aminobutylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 28:3-[4-(3-Aminopropylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 29:3-[3-(3-Aminopropylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 30:3-[4-(4-Aminobutylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Compound 31:4-[3-(4-Aminobutylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 32:4-[4-(3-Aminopropylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 33:4-[3-(3-Aminopropylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 34:4-[4-(4-Aminobutylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 35: 5-[3-(4-Aminobutylamino)-propylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 36: 5-[4-(3-Aminopropylamino)-butylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 37: 5-[3-(3-Aminopropylamino)-propylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 38: 5-[4-(4-Aminobutylamino)-butylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtha-[2,3-d][1,3]dioxol-5-yl]-amide

Compound 55: 5-[(2-Dimethylaminoethyl)-methylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 56:4-Amino-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide

Compound 57: 5-Aminopentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 58:3-(5-Aminopentylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide

Urea Series:

Compound 39:1-(4-Aminobutyl)-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 40:1-[4-(3-Aminopropylamino)-butyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 41:1-[3-(4-Aminobutylamino)-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 42:1-[3-(3-Aminopropylamino)-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 43:1-[4-(4-Aminobutylamino)-butyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 44:1-{2-[3-(4-Aminobutylamino)-propylamino]-ethyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 45:1-{2-[4-(3-Aminopropylamino)-butylamino]-ethyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 46:1-{4-[4-(4-Aminobutylamino)-butylamino]-butyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 47:1-{3-[3-(3-Aminopropylamino)-propylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 48:1-{3-[4-(3-Aminopropylamino)-butylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 49:1-[2-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-ethyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 50:1-[3-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 64:1-[4-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-butyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 65:1-(5-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-pentyl)-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 66:1-{3-[3-(4-Aminobutylamino)-propylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 67:1-{4-[3-(4-Aminobutylamino)-propylamino]-butyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Compound 68:1-{4-[4-(3-Aminopropylamino)-butylamino]-butyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

Sulfamido Series:

Compound 61: 2-(4-Aminopentylamino)-ethanesulfonic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 62: 2-[4-(4-Aminobutylamino)-butylamino]-ethanesulfonic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 63: 2-[3-(3-Aminopropylamino)-propylamino]-ethanesulfonic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 51: 2-(4-aminobutylamino)-ethanesulfonic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 52: 2-[3-(4-Aminobutylamino)-propylamino]-ethanesulfonic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 53: 2-[4-(3-Aminopropylamino)-butylamino]-ethanesulfonic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 54:2-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-ethanesulfonicacid3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 59:2-{3-[3-(3-Aminopropylamino)-propylamino]-propylamino}-ethanesulfonicacid3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

Compound 60:2-{4-[4-(4-Aminobutylamino)-butylamino]-butylamino}-ethanesulfonic acid3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide

and inorganic or organic acid addition salts thereof.

The compounds of the invention are, for example, the compounds of thegeneral formula 1 wherein:

-   -   R represents a hydrogen atom or C₁₋₄alkyl,    -   A represents CO(CH₂)_(n), CONH(CH₂)_(n), or SO₂(CH₂)_(n) where        n=2, 3, 4, or 5,    -   R1 represents a hydrogen atom or C₁₋₄alkyl,    -   R2 represents a hydrogen atom or C₁₋₄alkyl, or can also be        (CH₂)_(m)—NR3R4, where m=2, 3, 4, or 5,    -   R3 represents a hydrogen atom or C₁₋₄alkyl,    -   R4 represents a hydrogen atom or C₁₋₄alkyl, or can also be        (CH₂)_(p)—NR5R6, where p=2, 3, 4, or 5,    -   R5 represents a hydrogen atom or C₁₋₄alkyl, and    -   R6 represents a hydrogen atom or C₁₋₄alkyl, or can also be        (CH₂)_(q)—NH₂, where q=2, 3, 4, or 5.        with the exception of compounds wherein A=CO(CH₂)₂ or        A=SO₂(CH₂)₃ and R1=R2=H.

Advantageously, R1=H, R3=H, and R5=H. R can also represent preferably ahydrogen atom.

The compounds of the invention are, for example, compounds of thegeneral formula 1 wherein A represents CO(CH₂)_(n) or CONH(CH₂)_(n)where n=2, 3, 4, or 5, with the exception of compounds whereinA=CO(CH₂)₂ and R1=R2=H.

The compounds of the invention are, for example, compounds of thegeneral formula 1 such as defined above wherein R=H.

The compounds of the invention are, for example, compounds of thegeneral formula 1, such as defined above, wherein R1 and R2 are notsimultaneously H when R=H and A=CO(CH₂)_(n) where n=2, 3, or 4.

One particular embodiment of the invention relates to compounds of thegeneral formula 1, such as defined above, wherein R2=(CH₂)_(m)—NR3R4,preferably R4=(CH₂)_(p)—NR5R6, and in particular m=3 or 4, and p=3 or 4,for example compounds of the general formula 1, wherein R6=H, C₁₋₄alkyl,or else (CH₂)_(q)—NH₂, where q=3.

The present invention relates in particular to compounds of the formula1 selected from the group consisting of compounds 1 to 50, 55 to 58, and64 to 68 described hereabove, and inorganic or organic acid additionsalts thereof.

More particularly, the compounds of the invention may be selected fromthe group consisting of compounds 14 to 50, and 64 to 68 such as definedabove, and inorganic or organic acid addition salts thereof.

For example, the compounds of the invention can be selected from thegroup consisting of compounds 14, 16 to 18, 21 to 24, 27, 28, 31 to 36,39 to 41, 44 to 50, 54, 64 to 68, such as defined above, and inorganicor organic acid addition salts thereof.

The isomeric compounds according to the invention are within the scopeof the invention.

In the present invention, <<pharmaceutically acceptable>> as used hereinmeans what is useful in the preparation of a pharmaceutical compositionwhich is generally safe, non toxic and which is not biologically orotherwise undesirable, and which is convenient for both veterinary andhuman pharmaceutical use.

As used herein, the term <<pharmaceutically acceptable salts>> of acompound means salts which are pharmaceutically acceptable, as definedherein, and which have the desired pharmacological activity of theparent compound. Within the scope of the present invention, it is meantmore particularly addition salts of pharmaceutically acceptableinorganic or organic acids.

Pharmaceutically acceptable acids include, but are not limited to,hydrochloric, hydrobromic, sulfuric, phosphoric, acetic,trifluoroacetic, lactic, pyruvic, malonic, succinic, glutaric, fumaric,tartaric, maleic, citric, ascorbic, oxalic, methanesulfonic, camphoric,and sulfamic acids. The compounds according to the invention arecharacterized in that they are water soluble by forming inorganic ororganic salts, together with the basic nitrogen atoms of the side chainin the 4-position.

A further object of the present invention is the use of compounds of theformula 1, for anticancer treatment of liquid tumors and solid tumors,such as melanomas, colorectal cancers, lung cancers, prostate cancers,bladder cancers, breast cancers, uterus cancers, esophageal cancers,stomach cancers, pancreas cancers, liver cancers, ovarian cancers,leukemias in particular lymphomas and myelomas, ENT cancers and braincancers.

These compounds can be used in combination with other anticancertreatments, which may be cytotoxic or cytostatic, such as platinumderivatives, taxans, vincas, 5-FU, to increase their therapeuticeffectiveness for the purpose of treating tumors resistant to usualtherapies.

Another object of the present invention is a process for preparing thesecompounds. This process involves the podophyllotoxin of the formula 2 asa raw material. In particular the demethylation reaction of thepodophyllotoxin by the reactant pair methionine (ordimethylsulfide)-methanesulfonic acid, or in the presence oftrifluoroacetic acid or acetone, and water, is used according to themethod described in French patent FR 2 742 439, to yield the4′-demethylepipodophyllotoxin of the formula 3. This compound can besubjected to a Ritter reaction in the presence of sulfuric acid or otherstrong acid, with an organic nitrile of the formula Ra—CN whereRa=—(CH₂)_(n)—X or —CH═CH₂, where n=3, 4, or 5 and X represents ahalogen atom, such as a chlorine atom, to obtain a compound of theformula 4. The organic nitrile may be in particular chloroacetonitrile,or more generally a halogenoalkylonitrile of the formula NC—(CH₂)_(n)—X.

The intermediate amide of the formula 4a can thus be formed, wherein nis from 3 to 5. When acrylonitrile is reacted instead ofhalogenoakylonitriles, the vinylamide intermediate of the formula 4b isobtained.

The amide intermediate of the formula 4a wherein n=1, and X=Cl, which isa known intermediate, is treated with thiourea in refluxing acetic acidto provide with an excellent yield the4β-amino-4-deoxy-4′-demethylpodophyllotoxin, a compound of the formula 6wherein R=H, according to the method described in patent application WO2007/010007.

The amide compounds of the formula 1 wherein A=(CH₂)_(n)—X and R=H areprepared as follows:

The intermediates of the formula 4 (4a or 4b) can be subjected to analkylation with an amine (monoamine, diamine or polyamine), inparticular putrescine, spermidine, or spermine, in a protected form.Polyamines have several amine functions, so that they should beprotected by protecting groups to leave a free primary amine position,for a good reaction selectivity. It is within the knowledge of theperson skilled in the art to select protecting groups such asbenzyloxycarbonyl, or t-butyloxycarbonyl groups to protect those aminefunctions which should be left unreacted.

For example, spermine protected by benzyloxycarbonyl (Z) ortertiary-butyloxycarbonyl (BOC) groups is described. Likewise spermidineprotected by Z or BOC groups is described.

Thus, the alkylation reaction will be carried out between a compound ofthe formula 4 and an amine of the formula HNR1R2a in a protected form,wherein:

-   -   R1 is as defined above,    -   R2a=C₁₋₄alkyl, an amine-protecting group, or (CH₂)_(m)—NR3aR4a,        where m is as defined above,    -   R3a=C₁₋₄alkyl, or an amine-protecting group,    -   R4a=C₁₋₄alkyl, an amine-protecting group, or (CH₂)_(p)—NR5aR6a,        where p is as defined above,    -   R5a=C₁₋₄alkyl, or an amine-protecting group,    -   R6a=C₁₋₄alkyl, an amine-protecting group, or (CH₂)_(q)—NR7a R8a,        where q is as defined above,    -   R7a=H or an amine-protecting group, and    -   R8a=an amine-protecting group.

Protecting amine functions is suitable to prevent the synthesis ofundesirable by-products, such that there is only one reactivity site,during the coupling reaction.

The following publications disclose the preparation of the variousamines with protecting groups: Protective Groups in Organic Synthesis(Th. W. Greene, 2^(nd) Ed, John Wiley and sons, 1991), or in Synthesis2002, 15, 2195; Bull. Chem. Soc. Jpn. 1998, 71, 699; Tet. Let. 1998, 39,439 and 443; Tet. Let. 2001, 42, 2709; OPPI 1994, 26, 599; Synthesis1994, 37; J. Org. Chem. 1998, 63, 9723; Tet. Let. 1994, 35, 2057, and2061, J. Med. Chem. 2004, 47, 6055; J. Med. Chem. 2003, 46, 5712; Tet.Let. 1995, 36, 9401; Tet. 2000, 56, 2449.

The amine-protecting groups may be in particular Z or BOC.Advantageously, all the protecting groups on the protected amine will beidentical.

The alkylation reaction between the protected amine and the compound ofthe formula 4 produces a compound of the formula 5 followed by acompound of the formula 7a after deprotection of the amine functionsprotected by amine-protecting groups (when such groups are present).

Thus, according to the set of selective protections by amine-protectinggroups, for example BOC or Z, those skilled in the art can prepare thecompounds of the formula 7a.

Optionally, the last step of the process according to the invention isthe deprotection of the amine functions protected by appropriate groups.

The resulting compounds will then be isolated from the reaction mixtureby techniques well known to those skilled in the art.

The compounds of the present invention contain chiral centers resultingfrom naturally occurring podophyllotoxin. In the compound of the formula2 (4′-DMEP), the hydrogen atoms in the 5, 5a, 8a, and 9 positions havethe following stereochemistry: H5α, H5aα, H8aβ, H9β. In the compound ofthe formula 3, the configuration of the asymmetric carbons isadvantageously the following: 5S, 5aS, 8aR, 9R.

The urea compounds of the formula 10 are prepared from the4β-chloroacetamido-4′-demethylpodophyllotoxin of the formula 4a (n=1,X=Cl), in which the 4′-phenol is protected with a hydroxyl-protectinggroup Y such as a benzyloxycarbonyl group. Treatment with the thioureaprovides the amino compound of the formula 8 with R=H, wherein the groupin the 4′-position is protected with a protecting group Y such as agroup Z (benzyloxycarbonyl), wherein the compounds of the formula 8 withR≠H can be formed according to a process disclosed in U.S. Pat. No.7,378,419.

This compound of the formula 8 (in particular wherein R=H) is thenreacted with isocyanates, such as halogenoalkylisocyanates of theformula O═C═N—(CH₂)_(n)—X, wherein X represents a halogen and n is achain having from 2 to 5 CH₂, to provide the compounds of the formula 9(according to the procedure disclosed in Heterocycles 1994, 39, 361).This intermediate of the formula 9 is reacted with the protected mono,di, tri, or tetramines (of the formula HNR1R2a) as mentioned above undertraditional alkylation conditions, i.e. in particular at roomtemperature in DMF in the presence of triethylamine and KI, to providecompounds of the formula 10a, followed by compounds of the formula 10bafter deprotection of the 4′-position on the podophyllotoxin backboneand of the protected amine functions.

The resulting compounds will then be isolated from the reaction mixtureby techniques well known to those skilled in the art.

The ureas can also be prepared with the compound of the formula 8 (inparticular wherein R=H), and phosgene or triphosgene to provide anon-isolated, activated carbonylated intermediate. This intermediatecorresponds to the following formula:

This intermediate is further reacted directly with a protected amine,diamine or polyamine of the formula H₂N—(CH₂)_(n)—NR1aR2a, wherein R1arepresents H, C₁₋₄alkyl or an amine-protecting group, and wherein R2aand n are as defined above (where R1a≠H when R2a=C₁₋₄alkyl or(CH₂)_(m)—NR3aR4a) to obtain a compound of the formula 10c, with theremaining synthesis being performed as described above (deprotection ofthe amine functions and of the phenol). The last deprotection stage,either in an acid medium, in the case of the group BOC, or by catalytichydrogenation in the case of the group Z, leads then to the freepolyamine compound of the general formula 1 wherein A=CONH(CH₂)_(n).

However, polyamine alkylation performed on podophyllotoxinhalogenoalkylamides is not a univocal reaction. The processingconditions of this classically used alkylation are an alkaline medium.It is important to conduct the reaction in a weakly alkaline medium,such as in the presence of triethylamine. The alkalinity of the mediumcan lead according to the procedure to a by-product resulting from theepimerization of the proton in the 2-position thus providing thecis-lactone derivative, of the formula 11, i.e. the isomer of theformula 1. A precise chromatography makes it possible however to isolatethe desired trans-lactone derivative. The following examples show analternative method to prevent such possible epimerization. This is byforming an alkanoic acid chain on the protected polyamine (compound ofthe formula 12 wherein R1a is as defined above), followed by couplingthe resulting product through peptide coupling with the4β-amino-4-deoxy-4′-demethylpodophyllotoxin of the formula 6 (inparticular wherein R=H), according to the reaction scheme below:

This peptide coupling is performed, advantageously in the presence ofTBTU, preferably with a polyamine protected with benzyloxycarbonylgroups, and having a propionic, butyric, or pentanoic moiety. The acidintermediates having a moiety with 2 carbons (formula 12, n=2) areprepared by condensation with methyl acrylate in a similar way to theproducts described in Tet. 2006, 62, 8332. The acid intermediates of theformula 12 wherein n=3 to 5 are prepared by an ordinary alkylation ofthe halogenoalkylester-protected amine which is then saponified intocarboxylic acid. The compounds of the formula 7b are subsequentlyobtained to provide the compounds of the formula 7 after deprotection ofthe protected amine functions.

The resulting compounds will then be isolated from the reaction mixtureby techniques well known to those skilled in the art.

The sulfonamide compounds are prepared as follows:

The compound of the formula 8 (in particular wherein R=H) is reactedwith chloroethyl sulfonyl chloride, to obtain the vinylsulfonamideintermediate, which is opposite to the various protected polyamines. Thedeprotections are performed by a traditional hydrogenolysis in thepresence of palladium on carbon, in the case of a protecting group Z, orin an acid medium in the case of the protecting group BOC.

The following non limiting examples illustrate the process techniquesused:

1—Preparation of Intermediates Intermediate I:4-amino-4′-demethyl-4-deoxypodophyllotoxin (Formula 6 wherein R=H)

This compound is prepared as described in patent application WO2007/010007.

Stage 1: Ritter reaction: Preparation of4β-chloroacetamido-4′-demethyl-4-deoxypodophyllotoxin of the formula 4a(wherein n=1 and X=Cl)

To a suspension of 30 g (0.075 mol) of 4′-demethylepipodophyllotoxin ofthe formula 3 in 47.5 mL (0.75 mol) of chloroacetonitrile, 0.5 mL ofconcentrated sulfuric acid is added dropwise at room temperature. Themixture is left under stirring at this temperature for 1 hour duringwhich period dissolution is observed followed with reprecipitation. 300mL of 2-propanol are added. The precipitate is filtered, rinsed with 200mL of 2-propanol and water up to pH=7. The resulting white solid isdried under vacuum at 40° C. to obtain 32.9 g of the chloroacetamidocompound of the formula 4a (n=1, X=Cl), i.e. 93% yield. Mp=240° C.

Stage 2: Preparation of 4-amino-4′-demethyl-4-deoxypodophyllotoxin(formula 6 wherein R=H)

A suspension of 17 g (0.0358 mol) of the4β-chloroacetamido-4′-demethyl-4-deoxypodophyllotoxin obtained above in75 mL of glacial acetic acid is heated to 80° C. with stirring. 4.2 g(0.0537 mol) of thiourea is added in one portion. The mixture is leftunder stirring at this temperature for 1 h 30, during which perioddissolution is observed followed with reprecipitation. The reactionmixture is filtered hot, rinsed with 75 mL of glacial acetic acid anddiisopropyl ether. The resulting white solid is dried under vacuum at40° C. to obtain 14.6 g of the compound of the formula 6, inhydrochloride form corresponding to a 93% molar yield. Mp >260° C.¹H-NMR (DMSO) δ 8.63 (m, 2H), 8.32 (m, 1H), 7.23 (s, 1H, H₈), 6.60 (s,1H, H₈), 6.18 (s, 2H, H_(2′), H_(6′)), 6.05 (d, 2H, J=2.1 Hz, OCH₂O),4.73 (d, 1H, J=4.5 Hz, H₄), 4.56 (d, 1H, J=5.2 Hz, H₁), 4.34 (m, 2H,H_(11a) and H_(11b)), 3.65 (dd, 1H, J=5.2 Hz, H₂), 3.62 (s, 6H, 2×OCH₃),3.06 (m, 1H, H₃).

Intermediate II: Preparation of4β-acrylamido-4′-demethyl-4-deoxypodophyllotoxin

To a suspension of 3 g (0.0075 mol) of 4′-demethylepipodophyllotoxin ofthe formula 3 in 10 mL of acrylonitrile, a few drops of concentratedsulfuric acid are added at room temperature. The mixture is left understirring at this temperature for 3 hours during which period dissolutionis observed followed with reprecipitation. 50 mL of 2-propanol areadded. The precipitate is filtered, rinsed with 2-propanol and water upto pH=7. The resulting white solid is dried under vacuum at 40° C. toobtain 2.64 g of the acrylamide compound. Mp=180° C. TLC SiO₂ (30:70heptane:AcOEt) Rf 0.25. Anal. C₂₄H₂₃NO₃, H₂O (MW=471.464): calc. C %61.14, H % 5.63, N % 2.66. found: C % 60.84, H % 5.34, N % 2.97.

Intermediate III: Preparation of4β-chlorobutyramido-4′-demethyl-4-deoxypodophyllotoxin

Stage 1: Preparation of 4β-chlorobutyramido-4-deoxypodophyllotoxin

This compound is prepared from podophyllotoxin and 4-chlorobutyronitrileaccording to the procedure disclosed in stage 1 of the preparation ofintermediate I, for the reaction of chloroacetonitrile with4′-demethylepipodophyllotoxin. TLC SiO₂ (9:1 CH₂Cl₂:Acetone) Rf=0.38,Yield=71%.

Stage 2: Preparation of4β-chlorobutyramido-4′-demethyl-4-deoxypodophyllotoxin (formula 4awherein n=3 and X=Cl))

4.46 g of the compound obtained in stage 1 above are suspended withstirring in 21.16 mL of methane sulfonic acid. 1.93 g of D,L-methionineis then added and the stirring is maintained for 2 hrs. The reactionmixture is poured into water with stirring and a precipitate is formed.Filtration and washing with water until neutral give, after drying anddewatering, 2.26 g (Yield=52%) of demethylation product. TLC SiO₂ (9:1CH₂Cl₂:Acetone) Rf=0.20. The product is used directly withoutpurification in the following alkylation steps.

Intermediate IV: Preparation of4β-bromopentanamido-4′-demethyl-4-deoxypodophyllotoxin

This compound is prepared in a similar way to the4β-chloroacetamido-4′-demethyl-4-deoxypodophyllotoxin, stage 1 ofintermediate I, but with the use of the corresponding reactant, i.e.5-bromobutyronitrile.4β-Bromopentanamido-4′-demethyl-4-deoxypodophyllotoxin is obtained with57% yield. TLC SiO₂ (95:5 CH₂Cl₂:MeOH) Rf 0.28. Spectrum characteristicsignals ¹H-NMR (DMSO) δ 5.39 (t, 2H, J=6.4 Hz, CH₂Br), 2.17 (t, 2H,J=7.2 Hz, CH₂CO), 1.79 (m, 2H, CH₂), 1.66 (m, 2H, CH₂).

2—Preparation of Compounds of the Invention Example 1 Preparation of3-dimethylamino-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamideor (4β-dimethylaminopropionamido)-4′-demethyl-4-deoxypodophyllotoxin)(Compound 5)

500 mg of 4β-amino-4′-demethyl-4-deoxypodophyllotoxin of the formula 6,and 146 mg of 3-dimethylaminopropionic acid are dissolved in 50 mL ofacetonitrile, together with 0.21 mL of triethylamine with stirring. 400mg of TBTU are added and stirring is continued for 6 h at roomtemperature. The reaction mixture is poured into water (300 mL) andextracted with ethyl acetate (3×100 mL). The organic phases are driedover Na₂SO₄, filtered and evaporated. The residue is flashchromatographed on SiO₂ (elution with 78:20:2 CH₂Cl₂:MeOH:NH₄OH). Afterevaporation, the residue is again chromatographed on preparative HPLC (XBridge OBD C18, 30×250 mm, 10μ) gradient elution CH₃CN/HCl 5 mM (from10/90 to 80/20). The fractions are extracted with ethyl acetate (2×100mL), dried, and evaporated. The residue is salified with HCl isopropanolin ethyl ether, filtered and dried to provide 246 mg of hydrochloride,as a white powder. Yield=37%. TLC SiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf0.38. NMR of the base: ¹H-NMR (DMSO) δ 8.36 (d, 1H, NH), 8.28 (s, 1H,OH), 6.76 (s, 1H, H₅), 6.53 (s, 1H, H₈), 6.24 (s, 2H, H_(2′), H_(6′)),5.99 (d, 2H, J=8.4 Hz, OCH₂O), 5.16 (dd, 1H, H₄), 4.50 (d, 1H, J=5 Hz,H₁), 4.25 (t, 1H, H_(11a)), 3.87 (t, 1H, H_(11b)), 3.62 (s, 6H, OMe),3.11 (dd, 1H, H₂), 2.93 (m, 1H, H₃), 2.42-2.55 (m, 2H, CH₂N), 2.24-2.33(m, 2H, CH₂N), 2.12 (s, 6H, NMe₂). Mass spectrum (APCI), m/z=499, M-H+.

Example 2 Preparation of4-dimethylamino-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramideor (4β-dimethylaminobutyramido)-4′-demethyl-4-desoxypodophyllotoxin)(Compound 6)

A solution of 570 mg of intermediate III obtained above is stirred for12 hrs in 25 mL of acetonitrile, together with 0.28 mL (5 eq.) ofdimethylamine. The reaction mixture is then poured onto ice and a 1N HClsolution is added to pH=4. Extraction is carried out with methylenechloride and then the aqueous phase is alkalinized with a NaHCO₃solution to pH=8. This phase is re-extracted with CH₂Cl₂, dried overNa₂SO₄, filtered and evaporated to yield 100 mg of an orange foam. Thehydrochloride is formed in methylethylcetone, by adding an HClisopropanol solution (3N). The hydrochloride is then filtered, washedwith methylethylketone, followed with ethyl ether. Once dried, thecrystals obtained represent 90 mg of an off-white powder. TLC SiO₂(90:10:1 CH₂Cl₂:MeOH:NH₄OH) Rf 0.47. Mp=169° C. ¹H-NMR (DMSO) δ 8.35 (d,1H, NH), 6.75 (s, 1H, H₅), 6.51 (s, 1H, H₈), 6.20 (s, 2H, H_(2′),H_(6′)), 5.96 (d, 2H, J=6.36 Hz, OCH₂O), 5.15 (dd, 1H, H₄), 4.47 (d, 1H,J=5 Hz, H₁), 4.26 (t, 1H, H_(11a)), 3.68 (t, 1H, H_(11b)), 3.59 (s, 6H,OMe), 3.34 (m, 2H, CH₂N), 3.08 (dd, 1H, H₂), 2.93 (m, 1H, H₃), 2.72 (s,6H, NMe₂), 2.22 (m, 2H, CH₂CO), 1.86 (m, 2H, CH₂). Anal. C₂₇H₃₃ClN₂O₈,calc. C % 55.43; H % 6.37; N % 6.06; found C % 55.74, H % 6.01, N %4.68.

Example 3 Preparation of3-[(2-dimethylaminoethyl)-methylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-propionamideor(4β[-3-[2-(N-methyl-N,N-dimethylamino-2-ethyl)]propionamide)]-4′-demethyl-4-desoxypodophyllotoxin)(Compound 3)

200 mg of intermediate II are dissolved in 20 mL of THF, and 1.15 mL ofN,N,N′-trimethylethylenediamine are introduced dropwise into thereaction mixture. The mixture is stirred for 12 h at room temperature,and then evaporated to dryness. At this stage a mixture of the 2 epimersin the 2-position (cis-lactone and trans-lactone) is obtained. A flashchromatography (elution with CH₂Cl₂:MeOH:NH₄OH 90:10-0.5) affords 70 mgof the 2-epimerized derivative (cis-lactone). Mp=178° C. ¹H-NMR (DMSO) δ8.41 (d, 1H, J=8.96 Hz, CONH), 8.29 (m, 1H, OH), 6.95 (s, 1H, H₈), 6.89(s, 1H, H₅), 6.42 (s, 2H, H_(2′), H_(6′)), 6.01 (d, 2H, J=4.04 Hz,OCH₂O), 5.08 (dd, 1H, J=6.6 Hz, H₄), 4.37 (s, 1H, H₁), 4.28 (t, 1H,J=9.2 Hz, H_(11a)), 4.01 (dd, 1H, J=4 Hz, J′=9.6, H_(11b)), 3.79 (dd,1H, J=1.6 Hz, J′=10.8, H₂), 3.69 (s, 6H, OMe), 3.32 (m, 3H, H₃, COCH₂),2.63-2.27 (m, 6H, CH₂N), 2.194 (s, 3H, NMe), 2.101 (s, 6H, NMe₂).

Example 4 Preparation of 5-dimethylaminopentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]-dioxol-5-yl]-amideor (4β-dimethylaminopentanamido-4′-demethyl-4-deoxy-podophyllotoxin)(Compound 7)

700 mg of brominated intermediate IV obtained above are stirred in 3.3mL of a 2M dimethylamine solution in THF, for 4 days, under nitrogenatmosphere. The medium is poured into ice and added with an HCl solution(0.1N) to pH=7. The medium is extracted with ethyl acetate to provideafter drying over Na₂SO₄, filtration, and evaporation 341 mg of an oilwhich is then purified by flash chromatography on SiO₂ (97:7:0.7CH₂Cl₂:MeOH:NH₄OH) to provide 200 mg of pure oil. The hydrochloride isformed by adding to the base dissolved in isopropanol a solution ofhydrochloric ethanol to acidic pH. TLC SiO₂ (90:10:1 CH₂Cl₂:MeOH:NH₄OH)Rf 0.23. Mp tacky=224° C. ¹H-NMR (DMSO) δ 8.38 (d, 1H, J=8.56 Hz, NH),6.77 (s, 1H, H₅), 6.53 (s, 1H, H₈), 6.23 (s, 2H, H_(2′), H_(6′)), 5.99(d, 2H, J=12.4 Hz, OCH₂O), 5.18 (dd, 1H, J=8.16 Hz, J′=4.76 Hz, H₄),4.49 (d, 1H, J=5.12 Hz, H₁), 4.29 (t, 1H, J=8 Hz, H_(11a)), 3.73 (t, 1H,J=10.34 Hz, H_(11b)), 3.63 (s, 6H, OMe), 3.22 (dd, 1H, J=5.16 Hz,J′=14.3 Hz, H₂), 3.3 (t, 2H, J=7.08 Hz, CH₂N), 2.93 (m, 1H, H₃), 2.71(s, 6H, NMe₂), 2.20 (t, 2H, J=6.88 Hz, CH₂CO), 1.59 (m, 4H, CH₂).

Example 5 Preparation of 5-[(2-dimethylaminoethyl)-methylamino]pentanoicacidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amideor4β[5-[2-(N-methyl-N-dimethylamino-2-ethyl)]pentanamide)]-4′-demethyl-4-deoxypodophyllotoxin(Compound 55)

This compound is prepared in the same way as the compound of Example 2,but using the brominated intermediate IV andN,N,N′-trimethylethylenediamine. The4β[5-[2-(N-methyl-N-dimethylamino-2-ethyl)]pentanamide)]-4′-demethyl-4-deoxy-podophyllotoxincontaminated with its 8a-epimer is obtained. A flash chromatography(eluting with CH₂Cl₂:MeOH:NH₄OH, 95:5:0.5 followed by 90:10-0.6) affordsto isolate the title compound. The dihydrochloride is crystallized fromisopropanol by adding hydrochloric ethanol. HPLC C8 Symmetry (Elutionwith 80 KH₂PO₄ Buffer at 3.4 g/L brought to pH=4 by addition of H₃PO₄/20CH₃CN). Retention time: 4.95 min. ¹H-NMR (DMSO) δ 8.38 (d, 1H, J=8.56Hz, NH), 6.78 (s, 1H, H₅), 6.53 (s, 1H, H₈), 6.24 (s, 2H, H₂, H_(6′)),6.00 (d, 2H, J=11.3 Hz, OCH₂O), 5.19 (dd, 1H, J=8.15 Hz, J′=4.6 Hz, H₄),4.50 (d, 1H, J=4.8 Hz, H₁), 4.29 (t, 1H, J=8 Hz, H_(11a)), 3.72 (dd, 1H,H_(11b)), 3.63 (s, 6H, OMe), 3.53 (m, 2H, CH₂N), 3.08-3.24 (m, 5H, CH₂N,H₂), 2.93 (m, 1H, H₃), 2.84 (s, 6H, NMe₂), 2.79 (s, 3H, NMe), 2.20 (t,2H, J=6.88 Hz, CH₂CO), 1.69 (t, 2H, CH₂), 1.57 (m, 2H, CH₂).

Example 6 Preparation of4-amino-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramideor (4β-aminobutanamido-4′-demethyl-4-deoxypodophyllotoxin) (Compound 56)

Stage 1:

To a solution of 1 g of 4β-amino-4′-demethyl-4-deoxypodophyllotoxin ofthe formula 6 in 50 mL of acetonitrile are added 510 mg of4-t-butoxycarbonylamino butyric acid (Bioorg. Med. Chem. Lett. 2005, 15,1969) with stirring, together with 0.40 mL of triethylamine. 800 mg ofTBTU are then added and stirring is continued at room temperature for 5hrs. The reaction mixture is poured into water, and extracted with ethylacetate. After evaporation of the solvent, the residue is purified byflash chromatography on SiO₂ (gradient elution from pure CH₂Cl₂ to90:9:1 CH₂Cl₂:MeOH:NH₄OH). Preparative chromatography on X Bridge C18,OBD, 30×250 mm, 10μ, eluent: gradient from 10:90 CH₃CN/H₂O to 90:10CH₃CN/H₂O, provides after evaporation of the pure fractions 460 mg of acolorless oil. Yield=31%. TLC SiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf 0.20,¹H-NMR (DMSO) δ 8.25 (s, 1H, OH), 8.22 (d, 1H, J=8 Hz, NH amide), 6.79(m, 1H, NH carbamate), 6.76 (s, 1H, H₅), 6.52 (s, 1H, H₈), 6.24 (s, 2H,H_(2′), H_(6′)), 6.00 (d, 2H, J=13.2 Hz, OCH₂O), 5.17 (dd, 1H, J=8 Hz,J′=4.4 Hz, H₄), 4.49 (d, 1H, J=4.8 Hz, H₁), 4.27 (t, 1H, J=8 Hz,H_(11a)), 3.74 (t, 1H, J=9.6 Hz, H_(11b)), 3.62 (s, 6H, OMe), 3.15 (dd,1H, J=14.4 Hz and J′=5.2 Hz, H2), 2.89-2.96 (m, 3H, H₃ and CH₂N), 2.13(t, 2H, J=7.2 Hz, CH₂CO), 1.62 (m, 2H, CH2), 1.36 (s, 9H, t-Bu).

Stage 2:

The carbamate intermediate obtained in stage 1 above is stirred at roomtemperature for 4 h in 25 mL of CH₂Cl₂, in the presence of 25 mL of HClisopropanol (3.3 M). After evaporation, a white precipitate is obtained,which is then filtered and washed with ethyl ether, and dried to yield275 mg of hydrochloride as a white powder. Yield 67%. Mp=284° C. TLCSiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf 0.18, MS (ESI+) m/z=485 (M-H+).¹H-NMR (DMSO) δ 8.44 (d, 1H, J=7.6 Hz, NH amide), 8.27 (m, 1H, OH), 7.91(m, 2H, NH₂ and HCl), 6.77 (s, 1H, H₅), 6.53 (s, 1H, H₈), 6.24 (s, 2H,H_(2′), H_(6′)), 6.00 (d, 2H, J=11.6 Hz, OCH₂O), 5.19 (d, 1H, J=4.4 Hz,H₄), 4.52 (d, 1H, J=5.2 Hz, H₁), 4.31 (t, 1H, J=8 Hz, H_(11a)), 3.74 (m,1H, H_(11b)), 3.63 (s, 6H, OMe), 3.17 (dd, 1H, J=14 Hz and J′=4.8 Hz,H₂), 2.95 (m, 1H, H₃), 2.81 (t, 2H, J=7.6 Hz, CH₂N), 2.27 (t, 2H, J=7.2Hz, CH₂CO), 1.83 (m, 2H, CH₂).

Example 7 Preparation of 5-aminopentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]-dioxol-5-yl]-amideor (4β-aminopentanamido-4′-demethyl-4-deoxypodophyllotoxin) (Compound57)

This compound is prepared in the same way as in Example 6 above, butusing 5-t-butoxycarbonylaminopentanoic acid.

Example 8

In the same way as in Example 6, but by using in place of4-t-butoxycarbonylaminobutyric acid the corresponding protecteddiamino-, triamino- or tetramino-acids having a propionic chain (whichare prepared with methyl acrylate, in an analogous way to thepublication Tetrahedron 2006, 62, 8335), compounds 8, 10, 12, 13, 14,29, 30, 27, 28, 58, 19, 20 and 18 of the formula 1 (whereinA=CO(CH₂)_(n), n=2) have been synthesized.

Example 9 Preparation of2-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}-ethanesulfonicacid3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amideor(4-β-2-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}-ethanesulfonamido-4′-demethyl-4-deoxypodophyllotoxin)(Compound 54) Stage 1: Preparation of4-β-vinylsulfonylamino-4′-benzyloxycarbonyl-4′-demethyl-4-deoxypodophyllotoxin

500 mg of4β-amino-4-deoxy-4′-benzyloxycarbonyl-4′-demethylepipodophyllotoxin ofthe formula 8 are dissolved in 20 mL of CH₂Cl₂ with 0.4 mL oftriethylamine. 0.1 mL of 2-chloroethanesulfonyl chloride in 5 mL ofCH₂Cl₂ is added dropwise with stirring at −15° C. Stirring is continuedfor 15 min and then the mixture is left to return to ambienttemperature, and stirring is continued for 4 hrs. The reaction mixtureis then poured into water and extracted with CH₂Cl₂. The organic phasesare combined, dried over Na₂SO₄, filtered and evaporated. The residue ispurified by flash chromatography, eluted with a gradient of pure heptaneto pure AcOEt. The pure fractions obtained are evaporated to yield 220mg of a foam. Yield=55%. TLC SiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf 0.7,¹H-NMR (DMSO) δ 8.03 (d, 1H, J=8.56 Hz, NH), 7.40 (m, 5H, Ar), 7.02 (dd,1H, J=16.36 Hz, J′=9.8 Hz, HC═), 6.91 (s, 1H, H₅), 6.53 (s, 1H, H₈),6.33 (s, 2H, H_(2′), H_(6′)), 6.15 (d, 1H, J=16.4 Hz, HC═), 6.09 (d, 1H,J=9.8 Hz, HC═), 6.01 (d, 2H, J=11.3 Hz, OCH₂O), 5.23 (s, 2H, CH₂Ph),4.67 (dd, 1H, J=8.24 Hz, J′=4.4 Hz, H₄), 4.59 (d, 1H, J=5.4 Hz, H₁),4.31 (t, 1H, J=8.04 Hz, H_(11a)), 4.13 (t, 1H, H_(11b)), 3.63 (s, 6H,OMe), 3.28 (dd, 1H, J=5.36 Hz and J′=18.48 Hz, H₂), 2.97 (m, 1H, H₃).

Stage 2: Addition of N1,N2,N3-tribenzyloxycarbonylspermine

220 mg of the vinyl derivative obtained from the above stage aredissolved in 10 mL of methanol. 220 mg ofN1,N2,N3-tribenzyloxycarbonylspermine are added to the reaction mixture,and stirring is continued for 5 days at room temperature. Afterevaporation under vacuum, water is added, and the mixture is extractedwith ethyl acetate. After drying of the organic phase, filtration andevaporation, a purification is carried out by flash chromatography(gradient elution from pure heptane to pure AcOEt and then to 90:9:1AcOEt:MeOH:NH₄OH). 70 mg of the tetraprotected addition compound isobtained, i.e. with a 16% yield (trans-lactone compound). Another2-epimer compound is also obtained (cis-lactone compound).

Analyses of the trans-lactone compound: TLC SiO₂ (90:9:1CH₂Cl₂:MeOH:NH₄OH) Rf 0.6. Analytical HPLC: X Bridge C8 4.6×250 mm, 5μ,eluent: 80:20 CH₃CN:H₂O—KH₂PO₄ 6.8 g/L pH=4, flow rate 1 mL/min,RRT=3.55 min. MS (ESI+) m/z=1094.

Stage 3: Hydrogenolysis of Protecting Groups

70 mg of the trans-lactone derivative obtained above are placed underhydrogen atmosphere, in a mixture of 10 mL of methanol and 5 mL ofCH₂Cl₂. 0.25 mL of HCl isopropanol are also added, together with 50 mgof 10% palladium on carbon. Vigorous stirring is continued for 5 hrs.The catalyst is filtered, washed with methanol, the residue isevaporated under vacuum and taken up with ethyl ether for crystallizingthe hydrochloride, which is filtered and dried under vacuum. 30 mg ofcrystals are obtained as hydrochloride (Yield 63%). Mp=191° C.Analytical HPLC: X Bridge C8 4.6×250 mm, 5 g, eluent: 15:85CH₃CN:H₂O—KH₂PO₄ 6.8 g/L pH=4, flow rate 1 mL/min, RRT=14.08 min. MS(ESI+) m/z=692 (M-H+).

Example 10

In a similar way to Example 9, compounds 51, 52, 53, 59, 60, 61, 62, and63 can be synthesized using the corresponding protected monoamines,diamines, triamines and tetramines, by condensation with the4-β-vinylsulfonylamino-4′-benzyloxycarbonyl-4′-demethyl-4-deoxypodophyllotoxinprepared in stage 1 of Example 9.

Example 11 Preparation of4-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramideor(4-β-4-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}-butyramido-4′-demethyl-4-deoxypodophyllotoxin)(Compound 21)

This compound is synthesized according to either of the following 2methods.

Method 1: Alkylation of the Chlorinated Derivative (Intermediate III):

This compound is prepared in a manner similar to Example 2. Thechlorinated intermediate III prepared above is used and condensed withtribenzyloxycarbonyl-spermine (described in Tet. Let. 1998, 39, 439) toprovide the4-β-4-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}-butyramido-4′-demethyl-4-deoxypodophyllotoxin,after hydrogenolysis according to the same method as in stage 3 ofExample 9.

Method 2: Peptide Coupling:

Stage 1:

7.51 g of tribenzyloxycarbonyl spermine (triZ-spermine) (Tet. Let. 1998,39, 439) are dissolved in 150 mL of acetonitrile with stirring. 2.1 mLof triethylamine are added followed with 2.25 g of methyl bromobutyrate,and then 900 mg of cesium carbonate. The reaction mixture is refluxedfor 20 hrs with stirring. The medium is poured into water and extractedwith ethyl acetate (3×200 mL), and the organic phases are dried overNa₂SO₄, filtered and evaporated. The residue is flash chromatographed onSiO₂ (gradient elution from pure CH₂Cl₂ to a mixture of 70% CH₂Cl₂ and30% of a 9:1 MeOH:NH₄OH mixture). 2.48 g of the mono alkylation ester ofthe spermine derivative are isolated: the methyl4-[3-(benzyloxycarbonyl-{4-[benzyloxycarbonyl-(3-benzyloxycarbonylaminopropyl)-amino]butyl}-amino)-propylamino]-butyrateof the formula 12a (as a methyl ester, wherein B=H),

TLC SiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf=0.4. Other chromatographyfractions contain the dialkylation derivative of the sperminederivative.

Stage 2:

2.48 g of this mono alkylated intermediate from Stage 1 are placed in 30mL of acetonitrile together with 0.45 mL of triethylamine. 0.55 mL ofbenzyl chloroformate in 5 mL of acetonitrile is added dropwise withstirring at room temperature, and the mixture is left under stirring for2 hrs. The reaction mixture is poured into water and extracted withethyl acetate, and the organic phases are dried over Na₂SO₄, filteredand evaporated. A flash chromatography is performed (gradient elutionfrom pure CH₂Cl₂ to a mixture consisting of 90% CH₂Cl₂ and 10% 9:1MeOH:NH₄OH). 0.95 g of the tetra protected spermine derivative isobtained: the methyl4-{benzyloxycarbonyl-[3-(benzyloxycarbonyl-{4-[benzyloxycarbonyl-(3-benzyloxycarbonylaminopropyl)-amino]-butyl}-amino)-propyl]-amino}-butyrate,corresponding to the formula 12a, wherein B=Z, as a colorless oil. Yield32%. TLC SiO₂ (90:10:1 CH₂Cl₂:MeOH:NH₄OH) Rf=0.79. ¹H-NMR (DMSO) δ 7.32(m, 21H, NH and 4Ph), 5.03 and 5.00 (2s, 8H, benzyl CH₂), 3.54 (m, 3H,OMe), 3.14 (m, 12H, CH₂N), 2.97 (m, 2H, CH₂N), 2.27 (m, 2H, CH₂CO),1.62-1.67 (m, 6H, CH₂), 1.37 (m, 2H, CH₂).

Stage 3:

The above ester (0.95 g) is placed under reflux with stirring in 60 mLof a 50:50 MeOH:water mixture and in the presence of 1.7 mL of 1N NaOHfor 1 hr. After cooling, it is acidified with 1N HCl to pH=2, andextracted with ethyl acetate, to yield the corresponding carboxylicacid: the4-{benzyloxycarbonyl-[3-(benzyloxycarbonyl-{4-[benzyloxycarbonyl-(3-benzyloxycarbonylaminopropyl)-amino]-butyl}-amino)-propyl]-amino}-butyricacid as a colorless oil, with a quantitative yield. TLC SiO₂ (95:5CH₂Cl₂:MeOH) Rf=0.32. ¹H-NMR (DMSO) δ 7.32 (m, 21H, NH and 4Ph), 4.99and 5.04 (2s, 8H, benzyl CH₂), 3.14 (m, 12H, CH₂N), 2.97 (m, 2H, CH₂N),2.15 (m, 2H, CH₂CO), 1.66 (m, 6H, CH₂), 1.37 (m, 4H, CH₂). ESI-MSm/z=825 M-H+.

Stage 4:

510 mg of 4β-amino-4′-demethyl-4-deoxypodophyllotoxin, the intermediateI obtained as mentioned above, are dissolved in 20 mL of acetonitrile inthe presence of 950 mg of the acid obtained from the above stage and0.34 mL of triethylamine. 370 mg of TBTU are added in one portion andstirring is continued at room temperature for 2 hrs. The reactionmixture is poured into water and extracted with ethyl acetate, theorganic solution is washed with b, dried, filtered, and evaporated. Theresidue is purified by flash chromatography (elution with 90:10CH₂Cl₂:MeOH) and then by preparative HPLC (X Bridge, C18, 10 OBD, 30×250mm), gradient elution CH₃CN:H₂O from 10:90 to 50:50. 430 mg (Yield 30%)of the protected spermine podophyllotoxin derivative are obtained.¹H-NMR (DMSO) δ 8.23 (d, 1H, J=8.16 Hz, NH), 7.32 (m, 4 Ph), 6.78 (s,1H, H₅), 6.52 (s, 1H, H₈), 6.23 (s, 2H, H_(2′), H_(6′)), 5.98 (d, 2H,J=17.08 Hz, OCH₂O), 5.19 (dd, 1H, H₄), 4.99 and 5.03 (2s, 8H, CH₂benzyl), 4.49 (d, 1H, J=4.8 Hz, H₁), 4.29 (t, 1H, J=7.2 Hz, H_(11a)),3.73 (m, 1H, H_(11b)), 3.62 (s, 6H, OMe), 3.15 (m, 13H, CH₂N and H₂),2.96 (m, 3H, CH₂N and H₃), 2.10 (m, 2H, CH₂CO), 1.61-1.68 (m, 6H, CH₂),1.37 (m, 4H, CH₂).

Stage 5:

The thus purified tetrabenzyloxycarbonylated compound (430 mg) isdissolved in a mixture of methanol (20 mL) and CH₂Cl₂ (10 mL). 5equivalents of an HCl isopropanol solution are added. The medium isplaced under hydrogen atmosphere in the presence of 50 mg of 10%palladium on carbon under vigorous stirring for 8 hrs. The catalyst isfiltered, rinsed with methanol, and then the filtrate is evaporated. Theresidue is chromatographed on preparative HPLC (column Xbridge C18, 10μ,OBD, 30×250 mm) eluting with a 5 mM HCl solution. The fractionscontaining the compound are freeze dried, to obtain 115 mg of a whitesolid. Mp=229° C. Analytical purity: 98.25% (anal HPLC Xbridge C8,elution with 15:85 CH₃CN:H₂O—KHPO₄ 6.8 g/l at pH=4).

¹H-NMR (DMSO) δ 8.47 (d, 1H, J=8.28 Hz, NH), 6.78 (s, 1H, H₅), 6.53 (s,1H, H₈), 6.23 (s, 2H, H_(2′), H_(6′)), 6.00 (d, 2H, J=10.2 Hz, OCH₂O),5.19 (dd, 1H, J=7.72 Hz, J′=4.68 Hz, H₄), 4.49 (d, 1H, J=4.96 Hz, H₁),4.29 (t, 1H, J=7.8 Hz, H_(11a)), 3.75 (t, 1H, J=9.8 Hz, H_(11b)), 3.62(s, 6H, OMe), 3.24 (dd, 2H, J=14.28 Hz, J′=4.8 Hz, H₂), 2.89-3.00 (m,15H, H₃ and CH₂N), 2.29 (m, 2H, CH₂CO), 1.88-2.08 (m, 6H, CH₂), 1.73 (m,4H, CH₂).

Example 12

In the same manner as described in Example 11, following Method 2 bypeptide coupling, the following compounds of the formula 1 are prepared(wherein A=CO(CH₂)_(n), n=3, 4, or 5): 2, 4, 6, 15, 16, 17, 33, 34, 32,31, 37, 38, 35, 36, 22, 23, 25, 26, 24, 9, 11, 55, and 56, using thecorresponding protected monoamines, diamines, triamines or tetramines,which have been grafted, such as indicated in Stage 1 of Example 11,Method 2, with ethyl bromobutyrate or ethyl bromopropionate. Theprotection (Stage 2), saponification (Stage 3), coupling (Stage 4) anddeprotection (Stage 5) stages are performed in the same manner.

Example 13 Preparation of1-{3-[4-(3-aminopropylamino)-butylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea(Compound 48) Stage 1: Preparation of4β-chloroacetamido-4′-demethyl-4′-benzyloxycarbonyl-4-deoxypodophyllotoxin

19.6 g of 4β-chloroacetamido-4′-demethyl-4-deoxypodophyllotoxin of theformula 4a (X=Cl, n=1) prepared in stage 1 of intermediate I, aredissolved in 400 mL of THF then 10 mL of pyridine are added. 6.5 mL ofbenzyl chloroformate dissolved in 50 mL of THF are then added withstirring at room temperature. The reaction mixture is stirred at ambienttemperature for 6 hrs. At the end of the reaction, the solution ispoured into 300 mL of 1N HCl then extracted with ethyl acetate (2×200mL). The organic phases are dried over Na₂SO₄, filtered and evaporatedto provide 27.9 g of the intermediate crude compound. TLC (95:5CH₂Cl₂:MeOH) Rf=0.29. This intermediate is used directly in Stage 2.

Stage 2: Preparation of4β-amino-4′-demethyl-4′-benzyloxycarbonyl-4-deoxypodophyllotoxin

27.9 g of the intermediate prepared in Stage 1, above, are dissolved in120 mL of dimethylacetamide, 24 mL of acetic acid and 24 mL of water.This is heated to 80° C. with stirring. At this stage thiourea is added(4.81 g), and the reaction is maintained at this temperature for 12 hrs.After cooling, the reaction mixture is poured slowly into a saturatedsolution of NaHCO₃ (500 mL). It is then extracted with ethyl acetate(200 mL) and the organic phases are washed with a saturated solution ofNaHCO₃ and then with a saturated solution of NaCl. The organic phasesare separated, dried over Na₂SO₄, filtered and evaporated. The residueis flash chromatographed (gradient pure heptane, CH₂Cl₂ to 90:10CH₂Cl₂:MeOH) to yield 13.8 g of4β-amino-4′-demethyl-4′-benzyloxycarbonyl-4-deoxypodophyllotoxin of theformula 8. Yield over the 2 stages=63%. TLC (95:5 CH₂Cl₂:MeOH) Rf 0.55.

Stage 3: Preparation of1-{3-[4-(3-tert-butoxycarbonylaminopropyl)-tert-butoxycarbonylaminobutyl]-tert-butoxycarbonylaminopropyl}-3β-[9-(4-benzyloxycarbonyloxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea

500 mg of the intermediate obtained in Stage 2 above are dissolved withstirring in 30 mL of CH₂Cl₂ together with 0.13 mL of triethylamine.Under stirring at 0° C., a solution of 100 mg of triphosgene in 20 mL ofCH₂Cl₂ is added under nitrogen atmosphere. After reaching roomtemperature, a solution of 500 mg of triBOC spermine (Tet. 2000, 56,2449) and 0.13 mL of triethylamine, in 20 mL of CH₂Cl₂, are addeddropwise. The reaction mixture is stirred at room temperature for 3 hrs.The reaction mixture is then poured into a saturated NaHCO₃ solution andthen extracted with CH₂Cl₂. The organic phases are dried over Na₂SO₄,filtered and evaporated. The residue is flash chromatographed on SiO₂(eluting with pure CH₂Cl₂ to 90:10 CH₂Cl₂:MeOH), to yield 370 mg of ayellow oil. Yield 37%. TLC SiO₂ (CH₂Cl₂:MeOH 90:10) Rf 0.65. ¹H-NMR(DMSO) δ 7.4 (s, 5H, arom. H), 6.81 (s, 1H, H₅), 6.53 (s, 1H, H₈), 6.35(s, 2H, H_(2′), H_(6′)), 5.98 (d, 2H, J=7 Hz, OCH₂O), 5.23 (s, 2H,CH₂Ar), 5.03 (dd, 1H, H₄), 4.60 (d, 1H, J=5.2 Hz, H₁), 4.32 (t, 1H,J=7.6 Hz, H_(11a)), 3.81 (t, 1H, J=10 Hz, H_(11a)), 3.63 (s, 6H, OMe),2.87 (m, 14H, H₂, H₃, CH₂N), 1.56 (m, 2H, CH₂), 1.37 (m, 33H, CH₂, CH₃).

Stage 4: Preparation of1-{3-[4-(3-tert-butoxycarbonylaminopropyl)-tert-butoxycarbonylaminobutyl]-tert-butoxycarbonylaminopropyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]-dioxol-5-yl]-urea

740 mg of protected (triBOC and 4′O-benzyloxycarbonyl) intermediateobtained in Stage 3 above are dissolved in 20 mL of methanol with 100 mgof 10% palladium on carbon with stirring and under hydrogen atmospherefor 2 hrs, with vigorous stirring. The solution is filtered from thecatalyst, washed with MeOH and then evaporated to dryness. The residueis flash chromatographed on SiO₂ (gradient CH₂Cl₂ to 90:10 CH₂Cl₂:MeOH)followed with preparative HPLC (X Bridge, OBD, C18, 10μ, 30×250 mm),eluting with 20:80 CH₃CN:H₂O up to 100% CH₃CN. Extraction of thefractions containing ethyl acetate, drying over Na₂SO₄, filtration andevaporation, give 630 mg of1-{3-[4-(3-tert-butoxycarbonylaminopropyl)-tert-butoxycarbonylamino-butyl]-tert-butoxycarbonylaminopropyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-ureaas a colorless oil. Yield 97%. TLC SiO₂ (90:10:1 CH₂Cl₂:MeOH:NH₄OH) Rf0.58.

Stage 5: Preparation of1-{3-[4-(3-aminopropylamino)-butylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea

630 mg of the triBOC intermediate above are dissolved in 10 mL of HClisopropanol (3M) and then left under stirring for 6 hrs at roomtemperature. The medium is evaporated to dryness and then taken up withethanol. The hydrochloride precipitate formed is filtered, washed withabsolute ethanol and with ethyl ether. 391 mg of salt is obtained, i.e.a 78% yield. TLC SiO₂ (40:40:20 CH₂Cl₂:MeOH:NH₄OH) Rf=0.55. Mp=166° C.HPLC Purity 97% (analytical HPLC X Bridge, 15:85 CH₃CN:H₂O:6.8 g/lKH₂PO₄— pH=4, RT=8.08). ESI-MS, m/z=628 (M-H+). Anal. C₃₂H₄₅N₅O₈, 3HCl,4.4H₂O=813.39 calc. C %, 52.14; H %, 6.56; N %, 9.50, found C %, 51.89,H %, 5.95, N % 9.58. ¹H-NMR (DMSO, D₂O) δ 6.82 (s, 1H, H₅), 6.52 (s, 1H,H₈), 6.24 (s, 2H, H₂, H_(6′)), 5.98 (d, 2H, J=10 Hz, OCH₂O), 5.01 (d,1H, J=4 Hz, H₄), 4.51 (d, 1H, J=4.8 Hz, H₁), 4.35 (t, 1H, J=8 Hz,H_(11a)), 3.84-3.94 (m, H_(11a), H₂O), 3.63 (s, 6H, OMe), 3.17 (m, 3H,H2, CH₂N), 2.95 (m, 11H, H3, CH₂N), 1.94 (m, 2H, CH₂), 1.76 (m, 2H,CH₂), 1.68 (m, 2H, CH₂).

Example 14

In the same manner as described in Example 13, compounds 39, 40, 41, 42,43, 47, and 48 can be prepared following the same procedure as describedin Stage 3, but using the corresponding protected diamines, triamines,and tetramines. The deprotection steps are performed as described inStage 4 with regard to hydrogenolysis of the groups Z, or as describedin Stage 5 with regard to cleavage of BOC groups.

Example 15

The urea compounds 44, 45, and 49 are prepared according to Example 2 byalkylation but using the protected triamines or tetramines in place ofdimethylamine, and the1-chloroethyl-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-ureaderivative (disclosed in Heterocycles 1994, 39, 361) in place ofintermediate III. The deprotection stages are performed in the samemanner as described in Example 13, in Stage 4 and Stage 5.

Example 16

The urea compounds 46, 47, 64, 65, 66, 67, and 68 are prepared accordingto the procedure described in Stage 3 of Example 13, but using the3-chloropropylisocyanate, the 4-chlorobutylisocyanate, the5-chloropentylsiocyanate (Bull. Soc. Chim. Fr. 1959, 611), in place oftriphosgene, to lead to the corresponding alkyl ureas. The followingstages are conducted as indicated in Example 15.

Example 17

Compound50:1-[3-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-ureais also prepared according to the principle of the process disclosed inExample 13, but using the appropriate reactant.

Stage 1: Preparation of(3-tert-butoxycarbonylaminopropyl)-[4-(tert-butoxycarbonyl-{3-[3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)propylamino]-propyl}-amino)-butyl]-carbamicacid tertiary-butyl ester

A solution of triBOC spermine (Tet. 2000, 56, 2449) (6 g, 11.2 mmol, 1eq) in 100 mL of acetonitrile is added with N-(3-bromopropyl)phthalimide(3 g, 11.2 mmol, 1 eq) and cesium carbonate (7.2 g, 22.4 mmol, 2 eq).The medium is refluxed with stirring for 8 hrs. After evaporation, it ispoured into water (400 mL) and extracted with AcOEt (3×200 mL). Theorganic phases are washed with a saturated aqueous NaCl solution,separated, dried over Na₂SO₄ and evaporated. The residue is flashchromatographed on SiO₂ and eluted with a gradient of pure CH₂Cl₂ toCH₂Cl₂:MeOH:NH₄OH (80:18:2) to yield after evaporation 2.31 g of acolorless oil (Yield 30%). TLC SiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf=0.5.MS: m/z=690 (M-H+).

Stage 2: Preparation of[3-(tert-butoxycarbonyl-{4-[tert-butoxycarbonyl-(3-tert-butoxycarbonylaminopropyl)-amino]-butyl}-amino)-propyl]-[3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-propyl]-carbamicacid tertiary-butyl ester

The compound obtained in Stage 1 above (2.31 g, 3.3 mmol, 1 eq) isdissolved in 50 mL of THF with stirring. A solution of BOC₂O (0.8 g, 3.7mmol, 1.1 eq) in 10 mL of THF is then added dropwise at roomtemperature. Stirring is continued for 4 hrs and then the medium ispoured into water and extracted with AcOEt (3×100 mL), dried (Na₂SO₄),filtered, and evaporated. The residue is flash chromatographed with agradient from pure heptane to pure AcOEt. 1.49 g (Yield 56%) is obtainedafter evaporation.

Stage 3: Preparation of(3-aminopropyl)-[3-(tert-butoxycarbonyl-{4-[tert-butoxycarbonyl-(3-tert-butoxycarbonylaminopropyl)-amino]-butyl}-amino)-propyl]carbamicacid tertiary-butyl ester (Aminopropyl tetraBOC spermine)

The compound from Stage 2 above (1.49 g, 1.88 mmol, 1 eq) is refluxed in50 mL of EtOH in the presence of hydrazine hydrate (0.5 mL, 16.1 mmol,8.5 eq) for 6 hrs. The cooled medium is filtered, washed with EtOH, andevaporated. The residue is flash chromatographed on SiO₂ (gradientelution pure CH₂Cl₂ up to 80:18:2 CH₂Cl₂:MeOH:NH₄OH). After evaporationof the pure fractions, 1.09 g of a colorless oil is obtained (yield88%). TLC SiO₂ (90:9:1 CH₂Cl₂:MeOH:NH₄OH) Rf=0.34. MS: m/z=660 (M-H+).

Stage 4: Coupling of aminopropyl tetraBOC spermine with4β-amino-4′-demethyl-4′-benzyloxycarbonyl-4-deoxypodophyllotoxin

The 4β-amino-4′-demethyl-4′-benzyloxycarbonyl-4-deoxypodophyllotoxinobtained in Stage 2 of Example 13 (0.88 g, 1.6 mmol, 1 eq) is dissolvedwith stirring in 100 mL of CH₃CN, together with 0.23 mL (1.6 mmol, 1 eq)of triethylamine, and cooled to 0° C. A solution of triphosgene (0.17 g,0.58 mmol, 0.35 eq) is then added dropwise. After reaching roomtemperature a solution of the mixture of the intermediate compoundaminopropyl tetraBOC spermine, obtained in Stage 3 above (1.09 g, 1.6mmol, 1 eq) and of 0.23 mL (1.6 mmol, 1 eq) of triethylamine in 30 mL ofCH₂Cl₂ is then added dropwise. After stirring for 3 hrs, the mixture ispoured into a NaHCO₃ solution, and extracted with CH₂Cl₂ (3×100 mL). Theorganic phases are separated, dried over Na₂SO₄, filtered and evaporatedto yield a residue which is purified by flash chromatography (gradientfrom pure CH₂Cl₂ to 90:10 CH₂Cl₂:MeOH). After evaporation 1.37 g (68%)of the protected urea derivative is obtained as a white foam. TLC SiO₂(95:5 CH₂Cl₂:MeOH) Rf=0.62. Analytic HPLC: column Xbridge C8, 5μ,4.6×250 mm, eluting with 80:20 CH₃CN:H₂O, RRT=7.7 min.

Stage 5: Deprotection of the 4′-position

The derivative obtained in Stage 4 (1.37 g) is hydrogenolysed with 50 mgof 10% palladium on carbon in 100 mL of MeOH under vigorous stirring for8 hrs. The catalyst is filtered and the filtrate is evaporated todryness. The residue is first purified by flash chromatography on SiO₂eluting with a gradient from pure heptane to pure AcOEt, and then byHPLC (X bridge C18 OBD 30×250 mm), with a gradient from 50:50 CH₃CN:H₂Oto pure CH₃CN. 0.95 g (yield=78%) of the compound deprotected in the4′-position is obtained. TLC SiO₂ (95:5 CH₂Cl₂:MeOH) Rf=0.33. AnalyticHPLC: column Xbridge C8, 5μ, 4.6×250 mm, eluting with 80:20 CH₃CN:H₂O,RRT=4.7 min

Stage 6: Preparation of1-[3-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-ureavia deprotection of BOC groups

The compound obtained in Stage 5 above (0.95 g, 0.72 mmol) is dissolvedin 10 mL of CH₂Cl₂ in the presence of 10 mL of HCl-isopropanol (3M) withstirring for 4 hrs. The resulting precipitate is filtered and thenrinsed with Et₂O to yield 0.6 g (94%) of a white powder of1-[3-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtho[2,3-d][1,3]dioxol-5-yl]-urea.Mp=213° C. MS: m/z=685 (M-H+).

3—Pharmacological Test Example 18 In Vitro Pharmacological Tests

A cytotoxicity test is used. It measures the cell growth inhibition ofthe A549 human line (non-small cell lung cancer):

The A549 tumor cells are plated in a 96-well plate in RPMI 1640 mediumfree of phenol red (Seromed) to which is added 5% of fetal calf serum(100 μl/well, 1.25×10⁴ cells/ml). After incubation for 24 hrs at 37° C.in an incubator at 5% CO₂, the medium is replaced by that containing thecompound to be tested, followed by incubation of the plates for another48 hrs. Cell survival is assessed by measuring the luminescence aftersalting-out of the ATP from the medium using the cell lysis, luciferaseand luciferine solutions included in the ATP-lite-M™ kit following themanufacturer's guidelines (Packard, Rungis, France). Each experimentalcondition was tested at least three times in sixplicate.

The results show that the compounds of the invention have potentcytotoxic properties. The inhibiting concentration 50 (IC50), which isthe concentration of the compound to be tested providing a 50%inhibition of cell proliferation, is for example for compound 21:IC50=1.7×10⁻⁹ M, or for compound 48: IC50=1.2×10⁻⁸ M.

Example 19 In Vivo Pharmacological Tests

Experimental P388 tumor model. The model used is P388 murine leukemia(Tumor Models in Cancer Research. Teicher, B. A. ed., Humana Press Inc.,Totowa, N.J. Pp. 23-40, 2002), which is maintained by sequentialintraperitoneal transplantations in DBA/2 mice (DBA/2JIco mice, CharlesRiver), as previously disclosed (Classic in vivo cancer models: Threeexamples of mouse models used in experimental therapeutics. CurrentProtocols in Pharmacology Unit 5.24: 5.24.1-5.24.16, 2001).

The experiment is carried out according to a previously disclosedprotocol (Cancer Chemother. Pharmacol. 1998, 41, 437-447). Thiscomprises implanting 10⁶ P388 leukemia cells per mouse into C2DF1 hybridmice (CD2F1/CrlBR, Charles River, St Aubin-les-Elbeuf, France)intravenously at day zero. After the animals have been randomized intreatment and control cages, the compounds to be tested are administeredin one single injection by intraperitoneal route the day after the tumortransplantation, at day 1. The animals are then monitored every day,weighed twice per week, and any clinical reaction is recorded. Thesurvival rate is the parameter used to assess antitumor activity. Theincrease in survival rate is defined as the T/C_(survival) ratio (%),which corresponds to: (Median survival rate of the treated group/Mediansurvival rate of the control group)×100. The T/C_(survival) ratio iscalculated for each dosage and the largest value obtained represents themaximum increase in survival rate achieved (maximum activity), which isdefined as the optimum T/C_(survival) ratio.

The results show that the compounds have caused a significant increasein survival rate for animals with P388 leukemia.

As an example, compound 21 of Example 11 displays an optimumT/C_(survival) value of 186%, at a dose of 0.16 mg/kg, indicating thattreating the animals with this compound provided an 86% increase insurvival rate of the animals. In fact, according to the NCI's (NationalCancer Institute) criteria, a T/C_(survival) value is regarded assignificant if it is higher than at least 120% (Semin. Oncol. 1981, 8,349-361).

The loss in relative body weight of the animals, combined with theoptimum activity of the compounds, is highly lower than the toxicitythreshold, according to the NCI's criteria (Ann. Oncol. 1994, 5,415-422).

ABBREVIATIONS

APCI Atmospheric pressure chemical ionization

BOC tert-Butyloxycarbonyl

DMF Dimethylformamide

DMSO Dimethylsulfoxide

ESI Electro Spray Ionization

HPLC High Performance Liquid Chromatography

Mp Melting point

MS Mass Spectrum

NMR Nuclear Magnetic Resonance

Rf Frontal ratio

TBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate

THF Tetrahydrofuran

TLC Thin Layer Chromatography

Z Benzyloxycarbonyl

The invention claimed is:
 1. A method for treating cancer comprising theadministration to a person in need thereof of an effective amount of acompound of the general formula 1:

wherein: R represents hydrogen or C₁₋₄alkyl, A represents CO(CH₂)_(n),or CONH(CH₂)_(n), where n=2, 3, 4, or 5, R1=H, or C₁₋₄alkyl,R2=(CH₂)_(m)—NR3R4, where m=2, 3, 4, or 5, R3=H, or C₁₋₄alkyl R4=H,C₁₋₄alkyl, or (CH₂)_(p)—NR5R6, where p=2, 3, 4, or 5, R5=H, orC₁₋₄alkyl, and R6=H, C₁-C₄alkyl, or (CH₂)_(q)—NH₂, where q=2, 3, 4, or5, or a pharmaceutically acceptable salt thereof.
 2. The methodaccording to claim 1, wherein the cancer is selected from the groupconsisting of liquid tumors and solid tumors.
 3. The method according toclaim 2, wherein the solid tumor is selected from the group consistingof melanomas, colorectal cancers, lung cancers, prostate cancers,bladder cancers, breast cancers, uterus cancers, esophageal cancers,stomach cancers, pancreas cancers, liver cancers, ovarian, cancers,leukemias, ENT cancers and brain cancers.
 4. The method according toclaim 3, wherein leukemia is selected from the group consisting oflymphomas and myelomas.
 5. The method according to claim 1, wherein R=H.6. The method according to claim 1, wherein m=3 or 4, p=3 or 4, and q=3.7. The method according to claim 1, wherein the compound of formula 1 isselected from the following compounds: Compound 1:3-(2-Dimethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 2:4-(2-Dimethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 3:3-[(2-Dimethylaminoethyl)-methylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 4:4-[(2-Dimethylaminoethyl)-methylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 7: 5-dimethylaminopentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 8:3-(2-Diethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 9:4-(2-Diethylaminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 10:3-(2-Diethylaminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 11:4-(2-Diethylaminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 12:3-(2-Aminoethylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 13:3-(3-Aminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][,3]dioxol-5-yl]-propionamide,Compound 14:3-(4-Aminobutylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 15:4-(3-Aminopropylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 16:4-(4-Aminobutylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 17: 5-(4-aminobutylamino)pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 18:3-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 19:3-{3-[3-(3-Aminopropylamino)-propylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 20:3-{4-[4-(4-Aminobutylamino)-butylamino]-butylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 21:4-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 22:4-{3-[3-(3-Aminopropylamino)-propylamino]-propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 23:4-{4-[4-(4-Aminobutylamino)-butylamino]-butylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 24:5-{3-[4-(3-aminopropylamino)-butylamino]-propylamino}pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-55a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 25:5-{3-[3-(3-aminopropylamino)-propylamino]-propylamino}pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 26:5-{4-[4-(4-aminobutylamino)-butylamino]-butylamino}pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 27:3-[3-(4-Aminobutylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 28:3-[4-(3-Aminopropylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 29:3-[3-(3-Aminopropylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 30:3-[4-(4-Aminobutylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 31:4-[3-(4-Aminobutylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 32:4-[4-(3-Aminopropylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 33:4-[3-(3-Aminopropylamino)-propylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 34:4-[4-(4-Aminobutylamino)-butylamino]-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-butyramide,Compound 35: 5-[3-(4-Aminobutylamino)-propylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 36: 5-[4-(3-Aminopropylamino)-butylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 37: 5-[3-(3-Aminopropylamino)-propylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][,3]dioxol-5-yl]-amide,Compound 38: 5-[4-(4-Aminobutylamino)-butylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound40:1-[4-(3-Aminopropylamino)-butyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 41:1-[3-(4-Aminobutylamino)-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 42:1-[3-(3-Aminopropylamino)-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 43:1-[4-(4-Aminobutylamino)-butyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 44:1-{2-[3-(4-Aminobutylamino)-propylamino]-ethyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 45:1-{2-[4-(3-Aminopropylamino)-butylamino]-ethyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 46:1-{4-[4-(4-Aminobutylamino)-butylamino]-butyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 47:1-{3-[3-(3-Aminopropylamino)-propylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 48:1-{3-[4-(3-Aminopropylamino)-butylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 49:1-[2-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-ethyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound50:1-[3-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-propyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 55: 5-[(2-Dimethylaminoethyl)-methylamino]pentanoic acidN-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-amide,Compound 58:3-(5-Aminopentylamino)-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-propionamide,Compound 64:1-[4-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-butyl]-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 65:1-(5-{3-[4-(3-Aminopropylamino)-butylamino]-propylamino}-pentyl)-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 66:1-{3-[3-(4-Aminobutylamino)-propylamino]-propyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 67:1-{4-[3-(4-Aminobutylamino)-propylamino]-butyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,Compound 68:1-{4-[4-(3-Aminopropylamino)-butylamino]-butyl}-3-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl]-urea,and inorganic or organic acid addition salts thereof.
 8. The methodaccording to claim 1, wherein the compound of formula 1 is used incombination with other anticancer treatments.
 9. The method according toclaim 8, wherein the other anticancer treatments are cytotoxic orcytostatic.
 10. The method according to claim 8, wherein the otheranticancer treatments use platinum derivatives, taxans, vincas, or 5-FU.